High Performance Liquid Chromatography (HPLC)

 | Post date: 2022/05/30 | 
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). The sample is carried by a moving carrier gas stream of helium or nitrogen. HPLC has been used for the direct quantification of individual ecdysteroids in biological samples. This requires of course high sensitivity because of the low concentrations encountered and adequate sample clean up. 
Reversed-phase HPLC (RP-HPLC) is the most commonly used mode of HPLC and, as the name implies, this mode is just the reverse of normal phase HPLC (NP-HPLC), whereby the stationary phase is more nonpolar than the eluting solvent. Generally, RP-HPLC has a nonpolar stationary phase, e.g., C18 silica, and a moderately polar aqueous mobile phase. 
In RP-HPLC there is strong attraction between the polar solvent and polar molecules in the mixture being passed through the column, but there is not much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Therefore, polar molecules in the mixture spend most of their time moving with the solvent. Nonpolar compounds in the mixture tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They are less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules. They spend less time in solution in the solvent, and this slows them down on their way through the column, which means longer retention time. In RP-HPLC the polar molecules travel through the column more quickly.
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